Bacterial gene analysis has been revolutionized in recent years with the advent of high-throughput technologies that allow sequencing of entire genomes within a matter of months and comparative gene expression analyses of entire genomes within a couple of days. However, the genetic methods required to analyze specific genes identified by some of these high-throughput technologies in defined environments are by comparison slow, outdated and in many instances not even existing outside the better characterized species such as Escherichia coli. Furthermore, existing technologies, such a plasmid-based systems are not applicable in certain environments, for example animals and biofilms. In this proposal we seek funds to extend our laboratory's very successful program of genetic tool development by exploring the hypothesis that it is feasible to exploit the molecular properties of the site-specific transposon Tn7 to generate a kit of versatile, broad-host-range, site-specific gene integration vectors that can be used for diverse applications in different pathogenic bacteria. To test this hypothesis and to attain our goal we propose two specific aims. In aim 1 we will improve our prototype mini-Tn7 vectors by insertion of genetic transfer functions and transcriptional terminators, equip them with excisable antibiotic selection markers and reporter genes, and functionally analyze them in the genetically tractable model host Pseudomonas aeruginosa. In aim 2, the range of mini-Tn7 vectors will be extended to two other clinically significant respiratory pathogens, Burkholderia cenocepacia and Stenotrophomonas maltophilia, which are genetically less tractable and chromosomally more complex. The chromosomal insertion sites will be determined and chromosomally integrated lacZ fusions will be constructed and analyzed as proof-of-principle for the applicability of reporter gene analysis from single-copy, chromosomally located reporter genes in these bacteria. [unreadable] [unreadable]